Method and apparatus for detecting radiation

ABSTRACT

In analyzing radiation from a sample, single-quanta counting can be used to advantage especially at low levels of radiation energy, e.g. in the detection of fluorescent radiation. Preferred detection techniques include methods in which (i) fluorescence-stimulating radiation is intensity-modulated in accordance with a preselected code, (ii) wherein it is the fluorescent radiation which is intensity-modulated with the preselected code, and (iii) wherein modulation with a preselected code is applied to a sample to influence a property which functionally affects emitted fluorescent radiation. For registration of the signals from a sensing element of a single-photon detector, time of arrival is recorded, optionally in conjunction with registration of time intervals. Advantageously, in the interest of minimizing the number of pulses missed due to close temporal spacing of pulses, D-triggers can be included in counting circuitry.

This application is a divisional of Ser. No. 09/186,248, now U.S. Pat.No. 6,528,801, filed Nov. 04, 1998.

TECHNICAL FIELD

The invention is concerned with analytical technology and, morespecifically, with the detection of a fluorescent species or fluorophorein a sample.

BACKGROUND OF THE INVENTION

Fluorescent species or fluorophores emit fluorescent radiation whensuitably stimulated by stimulating radiation. The emitted radiation canbe used for chemical/biological analytic purposes, e.g. in determiningwhether a fluorophore of interest is present in a sample and inquantifying its concentration. One analytic technique of this type isdisclosed in U.S. Pat. No. 5,171,534 to Smith et al. wherein DNAfragments produced in DNA sequencing are characterized on the basis offluorescence of chromophores tagged to the fragments. Stimulatingelectromagnetic radiation may be monochromatic, or may includesignificant energy in a plurality of energy bands, e.g. as disclosed inU.S. Pat. No. 5,784,157 to Gorfinkel et al.

The stimulating radiation usually varies in time, either stochasticallyor regularly. Regular variation of the radiation intensity can beintroduced artificially by modulating the intensity of the radiationsource or the transmittance or reflectance of a filter element in theoptical path. Regularly modulated radiation may be termed as encodedradiation if the temporal variation of the radiation is used as acarrier of information. Associated with such encoded radiation is atemporal code, i.e. a time-domain function which corresponds to thetemporal evolution of the intensity of modulated radiation. Atime-domain function can be formed as a linear combination of severalsuitable functions whose respective contributions to the linearcombination can be quantified reliably. Suitable in this respect aresinusoidal functions of time, for example, oscillating at distinctfrequencies.

In prior-art techniques, the encoded radiation is considered ascontinuous, with the time dependence of detected radiation intensityregarded as a continuous time-domain function.

Further background includes several known single-photon detectiontechniques for which W. R. McCluney, Introduction to Radiometry andPhotometry, Artech House, 1996, pp. 114-122 provides a generalintroduction. Such techniques are designed for measuring modulatedradiation, and they can be classified into two groups: (a) asynchronousphoton counting and (b) synchronous detection. As described in AlanSmith, Selected Papers on Photon Counting Detectors, SPIE, Vol. MS 413,1998, methods (a) of asynchronous photon counting involve the detectionof a number of photons during a fixed time interval, e.g. one second,called the registration interval. These methods allow the determinationof an average frequency of photon arrival. This frequency varies intime, either stochastically or regularly, and synchronous counting canbe employed to measure the time variation. An essential limitation ofthis method is associated with the impossibility of measuringfrequencies of modulation that are higher than the repetition rate ofregistration intervals. This difficulty is inherent in the principle ofasynchronous counting, which is to keep track of the total number ofphotons received during the registration interval rather than registertheir times of arrival. A difficulty arises when the highest frequencyf_(mod) in the modulation spectrum of modulation radiation is comparableto or higher than the average frequency f_(phot) of single-photondetection. In this case, if the frequency limit is increased by reducingthe time interval chosen for counting, the technique becomesincreasingly inefficient because the counter will count nothing duringmost registration intervals.

Methods (b) of synchronous detection involve measurement of the time ofarrival of incident single photons. This time may be referenced to an“absolute” clock, or may be measured relative to or “synchronously with”a triggering excitation signal. The triggering signal may be associatedwith the arrival of the first of detected photons, for example. Suchmethods are particularly valuable for application to fast processes,e.g. the fluorescent decay of a single excited dye molecule asdescribed, e.g., by D. Y. Chen et al., “Single Molecule Detection inCapillary Electrophoresis: Molecular Shot Noise as a Fundamental Limitto Chemical Analysis”, Analytical Chemistry, Vol. 68 (1996), pp.690-696, typically requiring special electronics for handling fasttemporal variations. An essential limitation of these methods isassociated with the difficulty of maintaining records of high temporalresolution for a relatively long time. Thus, detecting photon arrivalsat the temporal resolution corresponding to nanosecond time intervalsover a one-second period requires acquisition of a billion data records.This makes methods of synchronous detection difficult to apply to thephotometry of relatively slowly varying modulated single-photon fluxes.

SUMMARY OF THE INVENTION

We have recognized that, in detecting a fluorescent species in a sample,single-photon counting can be used to advantage, especially at lowlevels of fluorescent signal energy. Preferred detection techniquesinclude methods in which (i) fluorescence-stimulating radiation isintensity-modulated in accordance with a preselected code, (ii) whereinit is the fluorescent radiation which is intensity-modulated with thepreselected code, and (iii) wherein modulation with a preselected codeis applied to a sample to influence a property, e.g. temperature,pressure, or an electric or magnetic field strength or frequency whichfunctionally affects emitted fluorescent radiation.

Preferably, for registration of the signals from a sensing element of asingle-photon detector, time of arrival is recorded, optionally inconjunction with registration of time intervals. Advantageously, in theinterest of minimizing the number of pulses missed due to close temporalspacing of pulses, D-triggers can be included in counting circuitry.

The preferred techniques are generally applicable to photometry oftime-encoded single-photon or particle fluxes. They involve measurementof time intervals between single-photon/particle arrivals combined withdata analysis that permits decoding of the encoded radiation, i.e.,discrimination between alternative possible codes and quantification ofdifferent combinations of mixtures of the codes. The techniques providefor the time intervals between successive pulses to be measuredasynchronously, without requiring an external clock reference or specialtriggering signal. They provide for efficient measurement and decodingof time-encoded single-photon or particle fluxes.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a schematic of a preferred first technique in accordance withthe invention, using a modulated light source.

FIG. 2 is a schematic of a preferred second technique in accordance withthe invention, using a dispersive element.

FIG. 3 is a schematic of a preferred third technique in accordance withthe invention, involving temporal encoding of different spectralcomponents of a fluorescent signal.

FIG. 4 is a schematic of a preferred fourth technique in accordance withthe invention, for registration of temporal parameters of a stochasticsequence of pulses of constant or similar shape.

FIG. 5 is a schematic of a preferred fifth technique in accordance withthe invention, wherein the fourth technique is integrated with themeasurement of time intervals.

FIG. 6 is a schematic of a preferred sixth technique in accordance withthe invention, wherein the fourth technique is augmented for furtherminimization of pulses lost to registration.

DETAILED DESCRIPTION

For purposes of the present description, no distinction need be madebetween “photon” and “quantum”, as each can result in a detector signal,typically an electrical signal or pulse for electronic processing inaccordance with techniques of the invention. Use of other types ofsignal processing is not precluded, e.g. by opto-electronic or purelyoptical means. It is understood that, in alternative processing means, adetector signal or a pulse being processed can be other than an electricsignal or pulse.

A. Single-Photon Detection in Methods for Fluorophore Identification

A special illumination technique is used, with a plurality of modulatednarrow-band sources, each modulated according to its own distinguishabletime-domain function. The narrow-band sources excite differentfluorophores differently, so that the emitted fluorescent radiation isencoded with information about the nature and composition of illuminatedfluorescent species. Photons are detected individually.

In a preferred first embodiment as illustrated by FIG. 1, a modulatedmulti-band light source producing encoded radiation of excitation offluorescence is combined with single-photon detection of encodedfluorescence signal.

FIG. 1 shows the light source 11 producing a radiation flux 12 which,via an optical illumination system 13, is incident on the container 14holding a fluorescent sample. The radiation flux 12 comprises aplurality of spectral bands, each modulated according to its owndistinguishable time-domain function. Fluorescent radiation 15 emittedby the fluorescent sample is received by an optical receiver system,e.g. an objective 16, and is directed to the optical input of asingle-photon detector 17. The output of the detector 17 is a stochasticstream 18 of electric pulses of similar shape, and information about theintensity of the received fluorescent radiation in a set time intervalis contained in the average frequency of the pulses arriving in theinterval. The temporal characteristics of the stream 18 of electricpulses are registered in a suitable form by the recorder 19 which isdescribed below in further detail, in connection with FIGS. 4 and 5. Ina preferred embodiment, the stochastic stream of pulses is characterizedin terms of the spacing in time between arrivals of successive pulses.The detection system may be complemented by communication means 120 fortransferring the recorded information at an appropriate rate from therecorder 19 to a signal processor unit 121.

A preferred second embodiment as illustrated by FIG. 2 can be viewed asan improvement over a known method for multicolor fluorescent detection,e.g. as disclosed in the above-referenced patent to Smith et al. In thistechnique, the fluorescent radiation emitted by an excited molecule isoptically analyzed into distinct wavelength channels, e.g. by a prism ora diffraction grating. The intensity of fluorescent radiation in each ofthe wavelength channels is then determined by photometric means. In thepreferred second embodiment, sensitivity is enhanced by the use ofsingle-photon detection.

FIG. 2 shows radiation 22 from a modulated optical source 21 beingfocused by a lens 23 onto a fluorescent sample 24. The modulated opticalsource 21 may produce one or several spectral bands that are modulatedeither together or independently with distinct time domain functions.Fluorescence 25 emitted by the sample 24 in response to the incidentradiation 22 is directed by an objective 26 to an optical processorwhich comprises a dispersive element 27, e.g. a prism or a diffractiongrating, and a set 29 of single photon detectors (SPD). The dispersiveelement 27 effects spectral analysis of the fluorescent signal.

Each of the SPD's produces at its output a stochastic stream ofelectrical pulses of similar shape, and information about the intensityof the received fluorescent radiation is contained in the temporalcharacteristics of the stochastic stream. With reference to FIG. 2, thetemporal characteristics 210 from each SPD are registered by a recorder211 whose structure is described below in further detail in connectionwith FIGS. 4 and 5. In a preferred embodiment, also described below infurther detail in connection with FIGS. 4 and 5, the description of thestochastic stream of pulses is specified in terms of the timeseparations between arrivals of successive pulses. The detection systemfurther comprises a signal processor unit 212 and means for transferringthe recorded information at an appropriate rate from the recorder 211 tothe signal processor unit 212.

FIG. 2 illustrates combination of a modulated light source forexcitation of fluorescence with a dispersive element for analyzing thefluorescent response into distinct spectral bands, and single-photondetection of modulated fluorescence in each of the spectral bands.Additionally, as in FIG. 1, the modulated light source can be multi-bandalso, so that the radiation flux 22 comprises a plurality of spectralbands, each modulated according to its own distinct time domainfunction. In this case, a preferred technique is advantageous further inthat different fluorescent species are distinguished both by theirfluorescence emission spectrum and their fluorescence excitationspectrum. This enhances the fidelity of fluorophore identification.

A preferred third embodiment of the invention, illustrated by FIG. 3,can be viewed as an improvement over a known technique for multicolorfluorescent detection, e.g. as applied according to the above-referencedpatent to Smith et al. The known technique is combined withsingle-photon detection, using a modulation technique disclosed in U.S.patent application Ser. No. 08/946,414, filed Oct. 7, 1997 by Gorfinkelet al. In accordance with the latter technique, radiation reflected,transmitted, or fluorescently emitted by an object is encoded in such away that the encoded radiation carries information about properties ofthe object, e.g. its color as characterized by reflected wavelengths, orthe identity and quantitative content of fluorescent species present inthe object. In the present embodiment of the invention, temporalencoding of different spectral components of a fluorescent signal iscombined with single-photon detection of the encoded spectralcomponents, for enhanced sensitivity.

FIG. 3 shows radiation 32 from optical source 31 being focused by anobjective 33 onto a fluorescent sample 34. In contrast to theembodiments illustrated by FIGS. 1 and 2, the optical source 31 need notbe modulated, and the radiation 32 may or may not be encoded.Fluorescence 35 emitted by the sample 34 in response to incidentradiation 32 is directed by an objective 36 onto an optical processorwhich comprises a dispersive element 37, e.g. a prism or a diffractiongrating, and a set of optical modulators 38. The dispersive element 37effects spectral analysis of the fluorescence 35. The spectralcomponents are directed onto a set of optical modulators 38 whichmodulate in time the resolved spectral components in such a way thateach different resolved spectral component is coded by a distinctfunction of time. The modulated components 39 of the fluorescentspectrum are combined by an optical element 310 into an optical flux 311focused onto the optical input of the single-photon detector 312. Theoutput of the detector 312 represents a stochastic stream 313 ofelectrical pulses of similar shape, whose temporal characteristics areregistered by the recorder 314 which is described below in further inconnection with FIGS. 4 and 5. In a preferred embodiment, also describedbelow in further detail, the description of the stochastic stream ofpulses is specified in terms of the temporal separation between arrivalsof successive pulses. The detection system further comprises means 315for transferring the recorded information at an appropriate rate to asignal processor unit 316.

B. Single Photon Detection of Modulated Photon Fluxes

A preferred fourth embodiment of the invention is illustrated by FIG. 4,of a method for registration of temporal parameters of a stochasticsequence of pulses of constant or similar shape.

The recorder of FIG. 4 operates with a controlled time resolution,controlled by a clock 45 which provides a regular sequence 46 ofelectrical pulses of constant shape which define the recording timeintervals. A stochastic stream 41 of electric input pulses may originatefrom a sensing element of a single-photon detector which is typically aphoto-multiplying tube (PMT) or an avalanche photo diode (APD).

The input pulses are not required to be of the same shape. With an APD,a special avalanche quenching circuit is used, either passive or active.Typically, the APD is pre-biased into its avalanche regime, for thefirst photon to initiate the avalanche. To prepare for the next photonarrival, the avalanche has to be quenched. It may be advantageous to usea so-called forced-quenching circuit which regularly quenches theavalanche condition, irrespective of whether an avalanche had actuallybeen initiated, so that the arrival of photons and the time of quenchingare not correlated. As a result, the avalanche-pulse duration will bestochastic also, depending on the time of photon arrival relative tosubsequent quenching.

The stream of pulses 41 is directed to an n-state cyclic state-shiftdevice or register 42. Such a device has n successive stable stateswhich may be numbered 0, 1, 2, . . . n−1, with a change from a state kto its successor state k+1 being triggered by an input pulse, and withstate n−1 having state 0 as its successor state. Between input pulses,the n-state cyclic state-shift device 42 retains its state. For example,for a 2-state cyclic state-shift device a flip-flop can be used, havinga sequence of stable states 0, 1, 0, 1, . . . , with each input pulsecausing a transition from 0 to 1 or from 1 to 0. It is not necessarythat the cyclic state-shift device return to its initial state when itsstate is read. This is in contrast to conventional photon counters whereeach reading of the counter data is accompanied by resetting the stateof the counter back to the initial state.

For the sake of specificity, without limiting the invention, a flip-flopwill be assumed in the following further description of FIG. 4. Theoutput from the flip-flop represents a stochastic sequence 43 ofrectangular pulses of variable length. The sequence 43 is directed to arecording device 44, which can be realized as an analog or digitalsignal recorder. The output signal 47 is transferred from the recordingdevice 44 to a signal processor (not shown).

The recorder of FIG. 4 operates essentially in an asynchronous mode.But, in contrast to asynchronous photon counters which record the totalnumber of photons arriving in a particular time interval, the preferredrecorder records their times of arrival. Accuracy of recording of thearrival time is controlled by the clock 45.

Time intervals are recorded without measuring the duration of theintervals. This function can be performed by one of a number of devicesknown to those skilled in the art, placed in an electrical circuitserially with the recorder and using its output signal 47. For example,a general-purpose computer can be used to process the array of dataacquired by the recording device 44.

In some applications it may be advantageous to integrate in a singledevice the functions of registering the time intervals betweensuccessive single photon detections and measurement of the timeintervals. Such an integrated preferred fifth embodiment of theinvention is illustrated by FIG. 5, for a stochastic stream of electricpulses 51 to which the shape and APD-quenching considerations concerningpulses 41 of FIG. 4 are applicable also.

As shown in FIG. 5, a stochastic stream of electric pulses 51 isdirected onto a flip-flop 52. Its output represents a stochasticsequence 53 of rectangular input pulses of variable length. The sequence53 is split three ways between counters 56 and 56′ and the controlleddelay line 531. The counter 56 receives the signal from the flip-flopdirectly, and the counter 56′ receives its signal through an inverter521. Thus, the counters 56 and 56′ are controlled by opposite-phasesignals. Instead of a flip-flop, 52, an n-state cyclic state-shiftdevice can be used, as described with reference to FIG. 4.Advantageously in this case, instead of two counters, 56 and 56′, up ton counters can be used.

A clock 54 provides a regular sequence 55 of electric pulses of constantshape which are counted by the counter 56. Exemplarily, counter 56 isthat counter whose input signal equals 1 at the time of clock pulsearrival. Advantageously, if the pulses 51 originate from and APD, theexternal quenching circuit which periodically forces the APD out of itsavalanche regime can be synchronized by the clock 54. There is noadvantage in increasing the quenching frequency beyond the clockfrequency which provides the basic discretization of time in thetechnique.

When a photon is detected and an electric pulse 51 enters the flip-flop52, one of the counters 56 and 56′ stops counting and the other beginscounting. The one counter that has just stopped counting then containsthe record 57 of how long the interval between two successive pulses haslasted, measured in terms of the number of clock cycles counted. Therecord 57 is transferred to the recording device 510 through acommutator 58 which serves to provide successive recording at intervalsof time so that, while one time interval is being recorded, the next oneis being measured. The commutator 58 is controlled by a switch signalwhich is derived by input signals 53 delayed by a characteristic time τ₁corresponding to the response time of the counter 56. The output of thecommutator 58 represents a sequence of codes 59 describing the measuredtime intervals between detected photons. The codes 59 appear at theoutput of the commutator 58 in stochastic fashion corresponding to thedetection of incoming photons and delayed by the time interval which isthe sum of τ₁ and the response time τ₂ of the commutator itself. It isadvantageous, therefore, to control the recording device 510 by switchsignals which are derived from the input signals 53, delayed from themoment of flip-flop switching by the time τ₁+τ₂. The output 514 of therecording device 510 represents the same sequence 59 of codes describingthe measured time intervals between detected photons. In contrast to thesequence 59, which is accumulated in time stochastically, the sequence514 can be transmitted in a regular fashion, e.g. at a constant rate,for further processing.

Further to the technique illustrated by FIG. 4, FIG. 6 illustratesinclusion of D-triggers for minimizing the number of pulses uncounteddue their close spacing in time. Electric pulses from a single-photondetector output are directed through a fast switch 61 to the input C ofa synchronous 8-bit binary counter 62. The result of the count is passedto the storage register 63 as an 8-bit word or byte. To avoid changingthe state of the counter 62 during storage, the synchronous pulsegenerator 65 shuts off the switch 61 simultaneously with sending a shortrecord pulse to the input Wr of the storage register 63. The output fromthe storage register 63 goes through the buffer 64 directly to theparallel port of a computer. Operational control error indicator isfacilitate by a logic comparator 66 equipped with an LED (light emittingdiode) 67. The parallel computer port is synchronized by a synchronouspulse through a delay line 68 with a suitable delay τ. The same delayedpulse synchronizes the logic comparator 66.

For an exemplary embodiment of the the technique illustrated by FIG. 6,the following may be specified and realized: a discretization frequencyof 125 KHz, a maximum number of pulses per discretization interval of256, a minimum time between registered pulses of 20 ns, a maximumaverage frequency of registered pulses of 32 MHz, and a maximum fractionof missed photons of 0.25%.

Techniques of the invention can be used to advantage in a variety ofapplications involving encoded electromagnetic radiation, includingmulticolor luminescent detection based on fluorescence spectroscopy andfluorescence excitation spectroscopy. They can be used in general sensorapplications with other modulated luminescence signals, e.g., thosebased on various spectroscopic techniques such as transmission,absorption, reflection, or Raman spectra, as well aselectro-luminescence, chemiluminescence and the like. The techniques areespecially useful for detecting weak signals, e.g. those prevalent inoptical communication links where signals are transmitted over longoptical fibers.

What is claimed is:
 1. A method for analyzing a sample by the detection of an chemiluminescence signal corresponding to radiation from the sample, comprising the steps of: (a) detecting successive quanta of an intensity-modulated chemiluminescence signal corresponding to radiation from the sample, with the modulation being over time in accordance with a preselected code; (b) determining time intervals between instances of detection of said quanta; (c) recording a sequence of said time intervals; and (d) comparing the recorded sequence with said code.
 2. The method according to claim 1, wherein the radiation from the sample is electromagnetic radiation.
 3. Apparatus for analyzing a sample by the detection of an chemiluminescencee signal corresponding to radiation from the sample, comprising: (a) a detector moiety for detecting successive quanta from an intensity-modulated chemiluminescence signal corresponding to radiation from the sample, with the modulation being over time in accordance with a preselected code; (b) a time-interval determination moiety operationally coupled to said detector moiety for determining time intervals between instances of detection of said quanta; (c) a recorder moiety operationally coupled to said time-interval determination moiety for recording a sequence of said time intervals; and (d) a comparator moiety operationally coupled to said recorder moiety for comparing the recorded sequence with said code.
 4. Apparatus for analyzing a sample by the detection of an chemiluminescence signal corresponding to radiation from the sample, comprising: (a) detector means for detecting successive quanta of an intensity-modulated chemiluminescence signal corresponding to radiation from the sample, with the modulation being over time in accordance with a preselected code; (b) time-interval determination means operationally coupled to said detector means for determining time intervals between instances of detection of said quanta; (c) recorder means operationally coupled to said time-interval determination means for recording a sequence of said time intervals; and (d) comparator means operationally coupled to said recorder means for comparing the recorded sequence with said code. 